Alveolar macrophage activation after trauma-hemorrhage and sepsis is dependent on NF-kappaB and MAPK/ERK mechanisms

The acute respiratory distress syndrome (ARDS) is a major cause of morbidity after injury. We hypothesized that alveolar macrophage (AMPhi) chemokine and cytokine release after hemorrhage and sepsis is regulated by NF-kappaB and MAPK. Adult male rats underwent soft tissue trauma and hemorrhagic shock (~90 min) followed by crystalloid resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) 20 h after resuscitation. AMPhi were harvested, and TNF-alpha, IL-6, and macrophage inflammatory protein (MIP)-2 release and serum IL-6 and TNF-alpha levels were measured at 5 h after HCLP. Lung tissues were analyzed for activation of NF-kappaB, myeloperoxidase activity, and wet/dry weight ratio. In control animals, AMPhi were stimulated with LPS with or without inhibitors of NF-kappaB and MAPK. Serum TNF-alpha and IL-6 levels and spontaneous AMPhi TNF-alpha and MIP-2 release were elevated (P < 0.05) after HCLP, concomitantly with the development of lung edema and leukocyte activation. Activation of NF-kappaB increased in lungs from the hemorrhage and CLP group compared with shams. Inhibition of NF-kappaB or the upstream MAPK significantly decreased LPS-stimulated AMPhi activation. Because enhanced release of inflammatory mediators by AMPhi may contribute to ARDS after severe trauma, inhibition of intracellular signaling pathways represents a target to attenuate organ injury under those conditions.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

Castration prevents suppression of MHC class II (Ia) expression on macrophages after trauma-hemorrhage.

Several studies indicate that cell-mediated immune responses, i.e., macrophage (MPhi) cytokine release capacities, myosin heavy chain (MHC) class II (Ia) expression, etc., are suppressed after trauma-hemorrhage in male mice. Testosterone has been shown to be responsible for the depression of MPhi cytokine responses in males after trauma-hemorrhage. Antigen presentation via MHC class II plays a key role in initiating and maintaining cell-mediated and humoral immune responses. It remains unknown, however, whether testosterone has any effect on MHC class II after trauma-hemorrhage. To study this, male C3H/HeN mice were castrated or sham castrated 2 wk before trauma (midline laparotomy) and hemorrhage (Hem; blood pressure 35 +/- 5 mmHg for 90 min and resuscitation) or sham operation. Four hours thereafter, MHC class II (Ia) expression was measured using flow cytometry. The results indicate that MHC class II (Ia) expression on peritoneal and splenic MPhi was significantly suppressed in male mice after trauma-hemorrhage. Prior castration, however, prevented the depression in MHC class II (Ia) expression on peritoneal and splenic MPhi after trauma-hemorrhage. Castration did not affect MHC class II (Ia) expression in MPhi from sham-castrated mice. Thus testosterone depresses MHC class II (Ia) expression on peritoneal and splenic MPhi after trauma-hemorrhage in males. Because MHC class II is necessary for an adequate immune response, our results suggest that depletion of male sex steroids or blockade of androgen receptors using agents such as flutamide might prevent immunosuppression via maintaining MHC class II (Ia) expression after trauma and severe blood loss.     

Insight into the mechanism by which metoclopramide improves immune functions after trauma-hemorrhage

Although studies have shownthat prolactin (Prl) and metoclopramide (Mcp) administration restoresthe depressed cell-mediated immune functions after hemorrhage, theunderlying mechanism responsible for the immunostimulatory effects ofMcp remains unknown. We hypothesized that Mcp improves immune responsesby upregulating the secretion of Prl. To test this hypothesis, maleC3H/HeN mice were subjected to sham operation or laparotomy (i.e., softtissue trauma) and hemorrhagic shock (Hem; 35 ± 5 mmHg for 90 min) and then resuscitated. Plasma Prl levels were determined 30 minafter Mcp (1 µg/g body wt sc at end of Hem) or vehicle (Veh)treatment in sham and Hem mice. The results indicate that plasma Prllevels increased significantly in Mcp-treated mice (sham-Veh 249.9 ± 5.3, Hem-Veh 229.9 ± 7.6, Hem-Mcp 596.9 ± 73.1 ng/ml,one-way ANOVA, P < 0.05 vs. Veh). To determine whetherMcp produces its salutary effects directly or indirectly via increasedPrl secretion, splenocyte proliferation and splenocyte interleukin(IL)-2 and IL-3 release from untreated sham or Hem mice were determinedin the presence of increasing concentrations of mouse Prl or Mcp. Theaddition of Mcp had no effect on splenocyte immune functions in vitro.However, the addition of Prl restored the hemorrhage-induced depressedsplenocyte proliferation as well as splenocyte IL-2 and IL-3 release invitro in a dose-dependent manner. Thus the beneficial effects of Mcp onimmune functions after Hem appear to be mediated by Prl. Because Mcpincreases plasma levels of the immunoenhancing hormone Prl, this agentshould be considered a useful adjunct for the treatment ofimmunodepression in trauma victims.

     

The female reproductive cycle is an important variable in the response to trauma-hemorrhage

Although immune functions in proestrus females are maintained after hemorrhage as opposed to decreased responses in males, it remains unknown whether such a sexual dimorphism also exists with regard to cardiovascular and hepatocellular functions under those conditions. To study this, male and female (estrus and proestrus) rats underwent a 5-cm midline laparotomy and were bled to and maintained at a mean blood pressure of 40 mmHg until 40% of the maximal bleed-out volume was returned in the form of Ringer lactate (RL). Rats were then resuscitated with four times the shed blood volume with RL. At 24 h thereafter, cardiac index; heart performance; hepatocellular function; and plasma estradiol, testosterone, and prolactin levels were measured. Cardiovascular and hepatocellular functions were depressed in males and estrus females (P < 0.05) but were not depressed in proestrus females after resuscitation. Plasma estradiol and prolactin levels were highest in proestrus females (P < 0.05), whereas males had high testosterone and the lowest estradiol levels (P < 0.05). Thus the female reproductive cycle is an important variable in the response to hemorrhage. Because low testosterone and high estradiol and prolactin levels appear to be beneficial for organ functions after trauma-hemorrhage, antagonism of testosterone receptors and/or increases in estradiol and prolactin levels in males and estrus females, respectively, may be novel approaches for improving organ functions under such conditions.     

Preinduction of heat shock proteins protects cardiac and hepatic functions following trauma and hemorrhage

Although studies have shown that induction of the heat shock proteins (HSPs), such as HSP-70, has various beneficial effects after ischemia-reperfusion, it remains unknown whether prior induction of HSP-70 has any salutary effects on cardiovascular and hepatocellular functions after trauma-hemorrhage and resuscitation. Male rats were exposed to heat stress (41 degrees C, 15 min) and then allowed to recover for 24 h at room temperature (21 degrees C). The rats then underwent laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of the maximal shed blood volume was returned in the form of Ringer lactate. Animals were then resuscitated with four times the volume of shed blood with Ringer lactate over 60 min. The maximal rate of the left ventricular pressure increase or decrease was measured up to 4 h after resuscitation. Cardiac output, hepatocellular function, plasma levels of tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were determined at 4 h after resuscitation. Cardiac and hepatic tissue were examined for HSP-70 by Western blot analysis. Left ventricular performance, cardiac output, and hepatocellular function decreased significantly following trauma-hemorrhage. Plasma levels of TNF-alpha and IL-6 were also significantly increased. However, prior heat stress attenuated cardiovascular and hepatocellular dysfunction, decreased circulating levels of proinflammatory cytokines following trauma-hemorrhage, and was associated with an increased abundance of HSP-70 in the heart and liver. Our data, therefore, suggest that preinduction of HSP-70 protects cardiovascular and hepatocellular functions following trauma-hemorrhage and resuscitation.     

Mechanism of salutary effects of estradiol on organ function after trauma-hemorrhage: upregulation of heme oxygenase

A growing body of evidence indicates that heme degradation products may counteract the deleterious consequences of hypoxia and/or ischemia-reperfusion injury. Because heme oxygenase (HO)-1 induction after adverse circulatory conditions is known to be protective, and because females in the proestrus cycle (with high estrogen) have better hepatic function and less hepatic damage than males after trauma-hemorrhage, we hypothesized that estrogen administration in males after trauma-hemorrhage will upregulate HO activity and protect the organs against dysfunction and injury. To test this hypothesis, male Sprague-Dawley rats underwent 5-cm laparotomy and hemorrhagic shock (35-40 mmHg for 93 +/- 2 min), followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. 17beta-Estradiol and/or the specific HO enzyme inhibitor chromium mesoporphyrin (CrMP) were administered at the end of resuscitation, and the animals were killed 24 h thereafter. Trauma-hemorrhage reduced cardiac output, myocardial contractility, and serum albumin levels. Portal pressure and serum alanine aminotransferase levels were markedly increased under those conditions. These parameters were significantly improved in the 17beta-estradiol-treated rats. Estradiol treatment also induced increased HO-1 mRNA expression, HO-1 protein levels, and HO enzymatic activity in cardiac and hepatic tissue compared with vehicle-treated trauma-hemorrhage rats. Administration of the HO inhibitor CrMP prevented the estradiol-induced attenuation of shock-induced organ dysfunction and damage. Thus the salutary effects of estradiol administration on organ function after trauma-hemorrhage are mediated in part via upregulation of HO-1 expression and activity.     

Corticosterone suppresses mesenteric lymph node T cells by inhibiting p38/ERK pathway and promotes bacterial translocation after alcohol and burn injury

Previous studies showed that alcohol (EtOH) intoxication before burn injury suppresses mesenteric lymph node (MLN) T cell functions and increases gut bacterial translocation. In this study, we examined whether corticosterone (Cort) plays any role in suppressing MLN T cell function and bacterial accumulation after EtOH intoxication and burn injury. Rats were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dl before receiving 25% total body surface area burn or sham injury. A group of rats was treated with the Cort synthesis inhibitor metyrapone (25 mg/kg) at the time of injury and on day 1 after injury. Two days after injury, a significant increase in blood Cort levels and suppression of MLN T cell proliferation and IL-2 production was observed in rats receiving combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. There was no change in T cell apoptosis after combined insult of EtOH and burn injury. Furthermore, T cell suppression was accompanied by a significant decrease in p38 and ERK1/2 activation (phosphorylation). There was no difference in JNK activation after EtOH and burn injury. Treatment of rats with metyrapone prevented the suppression of MLN T cell proliferation, IL-2 production, and p38 and ERK1/2 phosphorylation. Restoration of T cell function in metyrapone-treated animals was also associated with the decrease in bacterial accumulation in MLN. These findings suggest that EtOH intoxication before burn injury augments Cort release, which suppresses MLN T cell function by inhibiting p38 and ERK1/2 activation and promotes bacterial accumulation in MLN after EtOH and burn injury.